The nucleosome is the fundamental building block of eukaryotic chromosomes. Access to genetic information encoded in chromosomes is dependent upon where nucleosomes reside along the DNA. Alternative locations just a few nucleotides apart can have profound effects on gene expression. Yet the chromatin context in which most chromosomal and gene regulatory elements reside remains largely unknown. The work proposed here is expected to generate the highest resolution map of nucleosome locations throughout a genome. The Saccharomyces cerevisiae genome is chosen because of its simple genomic complexity and high degree of annotation, which will allow relationships between DNA regulatory elements and nucleosome positions to become particularly evident. Standard and post-translationally modified nucleosome core particles will be isolated and their locations throughout the genome mapped using three independent platforms: sequencing-based tagging, hybridization-based tiling arrays, and computationally-based comparative genomics that seeks to map nucleosome positioning sequences. These approaches are expected to reveal the fundamental aspects of chromatin architecture including DNA sequence determinants of positioning, rotational and translational settings of nucleosomal DNA, chromatin structure at specific classes of chromosomal elements, and the relationship between nucleosomal topology and promoter regulatory elements. This research is relevant to public health in that it provides an important foundation upon which gene regulation can be understood in animals. Mis-regulation of gene expression is a central cause of many human diseases, and thus a better understanding of gene regulatory mechanisms will provide more informed approaches in developing therapeutics. Nucleosomal control of gene expression is highly conserved among animals, thus lessons learned in flies and worms are directly applicable to human systems.